Methods to promote axonal regeneration have tremendous potential to treat the injured and diseased nervous system. This potential is most clear in the injured CNS, such as in spinal cord injury, where there is essentially no axon regeneration. Even in the periphery, increasing the speed and extent of axonal regeneration would provide important therapeutic benefits. In this proposal, we outline experiments to promote axon regeneration by therapeutically invoking the preconditioning response. The molecular basis of preconditioning is poorly understood but its ability to stimulate axonal regeneration after injury and to enhance axonal growth over non-permissive substrates makes it an important target for development of new approaches for treating the damaged nervous system. We have developed a fully in vitro preconditioning assay in primary neurons, which will allow for the first high throughput drug and genetic studies of this process. We plan to identify preconditioning pathways using high-throughput methods adapted from those we developed to explore axonal degeneration that enable rapid screening of compounds and genetic pathways. Using adult DRG neurons we plan to screen libraries of drug compounds and lentivirus open reading frame (ORF) libraries. First, we will optimize and miniaturize the screening assay and image analysis (R21 phase) and then use high-throughput screening and imaging analysis to identify compounds and/or genes that enhance axon re-growth by promoting a 'preconditioning' response (R33 phase). Second, we will develop secondary screens to assay molecular markers of preconditioning, neuronal sub-type-specific preconditioning responses, and a microfluidics based assay for axonal growth on inhibitory substrates (R21 phase). These assays will be used to further characterize 'hits' from the primary screens (R33 phase). Third, we will use a sciatic nerve crush assay to examine the in vivo activity of a few prioritized compounds identified in the screens (R33 phase). Through these experiments we hope to uncover agents and pathways of injury-induced preconditioning that will lead to new methods for stimulating robust axonal regrowth and growth on inhibitory substrates that will potentially lead to new treatments for the damaged nervous system.